In Vitro Development of Human iPSC-Derived Functional Neuronal Networks on Laser-Fabricated 3D Scaffolds

Abstract

Neural progenitor cells generated from human induced pluripotent stem cells (hiPSCs) are the forefront of ″brain-on-chip″ investigations. Viable and functional hiPSC-derived neuronal networks are shaping powerful in vitro models for evaluating the normal and abnormal formation of cortical circuits, understanding the underlying disease mechanisms, and investigating the response to drugs. They therefore represent a desirable instrument for both the scientific community and the pharmacological industry. However, culture conditions required for the full functional maturation of individual neurons and networks are still unidentified. It has been recognized that three-dimensional (3D) culture conditions can better emulate in vivo neuronal tissue development compared to 2D cultures and thus provide a more desirable in vitro approach. In this paper, we present the design and implementation of a 3D scaffold platform that supports and promotes intricate neuronal network development. 3D scaffolds were produced through direct laser writing by two-photon polymerization (2PP), a high-resolution 3D laser microstructuring technology, using the biocompatible and nondegradable photoreactive resin Dental LT Clear (DClear). Neurons developed and interconnected on a 3D environment shaped by vertically stacked scaffold layers. The developed networks could support different cell types. Starting at the day 50 of 3D culture, neuronal progenitor cells could develop into cortical projection neurons (CNPs) of all six layers, different types of inhibitory neurons, and glia. Additionally and in contrast to 2D conditions, 3D scaffolds supported the long-term culturing of neuronal networks over the course of 120 days. Network health and functionality were probed through calcium imaging, which revealed a strong spontaneous neuronal activity that combined individual and collective events. Taken together, our results highlight advanced microstructured 3D scaffolds as a reliable platform for the 3D in vitro modeling of neuronal functions.

Two-Photon Polymerization: Functionalized Microstructures, Micro-Resonators, and Bio-Scaffolds

“…Cultured neuronal networks are the subject of many biological studies because they allow researchers to investigate neuronal activity in a controlled environment, in comparison to in a live organism. These experiments provide, for instance, a better understanding of the primary disease mechanisms and investigating the response to medicines [ 153 ]. TPP can be employed to produce 3D scaffolds, with high-resolution, using biocompatible and nondegradable photoreactive resins [ 153 ].…”

“…These experiments provide, for instance, a better understanding of the primary disease mechanisms and investigating the response to medicines [ 153 ]. TPP can be employed to produce 3D scaffolds, with high-resolution, using biocompatible and nondegradable photoreactive resins [ 153 ]. Figure 18 shows examples of 3D scaffolds fabricated using Dental LT Clear (DClear) resin employed in 3D neural cell culture and their characterization.…”

“…Still, in Figure 18 b the scaffold design allows a noninvasive high-resolution visualization of real-time cell growth and neuronal network formation in a 3D environment. Immunofluorescence analysis showed that the fabricated scaffold provides a suitable 3D environment for the rapid development of neural stem cells (NSCs), facilitating the growth and long-term viability of neuronal networks ( Figure 18 c) [ 153 ], which may be related to the increased cell density in the cylindrical areas of the scaffolds that leads to strengthened neurons communications. Figure 18 d represents a quantitative ratio of young (DCX+) and mature (NeuN+) neurons as well as SOX2+ multipotent neural progenitor cells in the scaffold at day 25 of culture/differentiation.…”

“…In Figure 18 , the expression of young and mature neuronal markers DCX/MAP2/NeuN and SOX2 indicate a healthy formation of a neuronal network. Finally, on day 25 of 3D culture, a large proportion of neurons already expressed the early-born cortical neuronal marker CTIP2 ( Figure 18 h) [ 153 ].…”

“…( h ) Differentiation of early-born CTIP2+ cortical neurons from neural stem cells. Reproduced with permission [ 153 ]. Copyright © 2021, American Chemical Society.…”